A new method for SMN1 and hybrid SMN gene analysis in spinal muscular atrophy using long-range PCR followed by sequencing

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by progressive loss of motor neurons in the spinal cord. Approximately 95% of SMA patients have a homozygous deletion of the survival motor neuron 1 (SMN1) gene, whereas 5% harbor compound heterozygous mutations such as an SMN1 deletion allele and an intragenic mutation in the other SMN1 allele. It is difficult to detect intragenic mutations in SMN1 because of the high degree of homology shared between SMN1 and SMN2. Current methods analyze a restricted region from exon 2a to exon 7 in SMN1. We propose a new, efficient long-range polymerase chain reaction (PCR) method for detecting intragenic mutations in SMN1 (exon 1-8) and hybrid SMN genes. We analyzed 20 unrelated SMA patients using SMN copy number analysis, and the new long-range PCR method followed by sequencing. We thus confirmed a novel mutation in SMN1 exon 1 (c.5C>T) in three patients with SMA type III who also had an SMN1 deletion allele. Moreover, we confirmed three hybrid SMN gene types in eight patients. We report a novel SMN1 mutation responsible for a relatively mild SMA phenotype and three hybrid SMN gene types in patients with SMA type III.